For a number of years, it has been known that various microorganisms exhibit biological activity so as to be useful to control plant diseases. Although progress has been made in the field of identifying and developing biological pesticides for controlling various plant diseases of agronomic and horticultural importance, most of the pesticides in use are still synthetic compounds. Many of these chemical fungicides are classified as carcinogens by the Environmental Protection Agency (EPA), are toxic to wildlife and other non-target species. In addition, pathogens may develop resistance to chemical pesticides (see, e.g., Schwinn et al., in: Advances In Plant Pathology: Phytophthora Infestans, The Cause of Late Blight of Potato, p. 244, Academic Press, San Diego, Calif., 1991).
Biological control offers an attractive alternative to synthetic chemical fungicides. Biopesticides (living organisms and the naturally produced compounds produced by these organisms) can be safer, more biodegradable, and less expensive to develop.
Bacilli are known to produce antifungal and antibacterial secondary metabolites (Korzybski et al. (1978) "Section C: Antibiotics isolated from the genus Bacillus (Bacillaceae)" in: Antibiotics-Origin, Nature and Properties, American Society for Microbiology, Washington, DC (1978) Vol III.) and by Berdy (CRC Handbook of Antibiotic Compounds, Vols. I-XIV, (CRC Press, Inc., Boca Raton, Fla. 1980-87). Compounds known to be produced by B. pumilis include micrococcin P, pumilin, and tetain.
Kawaguchi et al. 1981 (U.S. Pat. No. 4,250,170) isolated a novel water soluble antibiotic from Bacillus with activity against a broad range of gram positive and gram negative bacteria. Stabb et al. (1994) Applied Environ. Microbiol. 60:4404-4412 have identified certain Bacillus spp. (Bacillus spp. includes B. subtilis, B. cereus, B. mycoides, B. thuringiensis) strains that exhibit antifingal activity. These strains have been shown to produce zwittermicin-A and/or kanosamine (Milner et al., (1996) Appl. Environ. Microb. 62:3061-3066), two antibiotic agents that are effective against the soil borne disease damping off, caused by Phytophthora medicaginis, P. nicotianae, P. aphanidermatum or Sclerotinia minor (See Stabb et al., supra). Zwittermicin-A is a water soluble, acid stable linear aminopolyol molecule (see, He et al, (1994) Tetrahedron Lett. 35(16):2499-2502) with broad spectrum activity against many fungal and bacterial plant pathogens. Kanosamine (Milner et al., 1996) also inhibits a broad range of fungal plant pathogens and a few bacterial species.
U.S. Pat. No. 5,049,379 to Handelsman et al. describes how Zwittermicin-A producing B. cereus control damping off in alfalfa and soybeans. When the seed was coated with B. cereus ATCC 53522, the pathogenic activity of root rot fungus was inhibited. Similarly, application of spore-based formulations of certain B. cereus strains to soybean seeds or the soil surrounding the seeds has been shown to improve soybean yield at field sites. (See, Osburne et al. (1995) Am. Phytopathol. Soc. 79(6):551-556). Methods of applying biopesticides are well known in the art and include, for example, wettable powders, dry flowables, microencapsulation, and liquid formulations of the microbe, whole broth or antibiotic fractions from suitable cultures. (See e.g., U.S. Pat. No. 5,061,495 to Rossall or U.S. Pat. No. 5,049,379 to Handelsman).
Tsuno et al. (Takashi Tsuno, Chiharo Ikeda, Kei-ichi Numata, Koju Tomita, Masataka Konishi and Hiroshi Kawaguchi (1986) J. Antibiotics XXXIX(7):1001-1003) report on a new amino sugar antibiotic from B. pumilus with activity against a broad range of bacteria in vitro.
Leifert et al., J. Appl Bacteriol. 78:97-108 (1995), reported the production of anti-Botrytis and anti-Alternaria antibiotics by two Bacillus strains, B. subtilis CL27 and B. pumilis CL 45. The whole broth and cell-free filtrates were active against Botrytis and Alternaria in in vitro tests and were active against Botrytis in in vivo small plant tests on Astilbe. Leifert et al. (1997) U.S. Pat. No. 5,597,565 disclose B. subtilis, B. pumilis, and B. polymyxa that are particularly effective at inhibiting post harvest disease causing fungi, Alternaria brassicicola and Botrytis cinerea. They also disclose the presence of antibiotics produced in the cell-free culture filtrate and their activity at different pH values, but they do not identify these compounds. The compounds from B. subtilis lose activity at low pH, while the activity from the B. pumilus extracts occurs only at pH values below 5.6. Leifert et al. (1998) U.S. Pat. No. 5,780,080 discloses cabbages that can be treated with B subtilis, B pumilis, and B. polymyxa strains to inhibit Alternaria brassicicola and Botrytis cinerea
Loeffler et al. (1986) J. Phytopathology 115:204-213, disclose B. subtilis, B. pumilus, B. licheniformis, and B. coagulans strains that produce various antibiotics with antifungal and antibacterial activity. B. pumilus produced bacilysin and iturin A. Bacilysin is a very small compound with a molecular weight of 270, that inhibits only yeast. The iturins, which are soluble in polar solvents, have broad antifungal and antibacterial activity.
Rossall (1994) U.S. Pat. No. 5,344,647 discloses Bacillus subtilis strains with broad anti-fungal activity. Rossall's (1991) U.S. Pat. No. 5,061,495 provides a novel antibiotic from B. subtilis that is 63,500 Dalton, precipitates at a pH below 5 and has activity against gram positive bacteria and fungi (Botrytis and Erysiphe). Sholberg et al. (1995) Can. J Microbiol. 41:247-252, Swinburne et al. (1975) Trans. Brit. Mycol. Soc. 65:211-217, Singh and Deverall, (1984) Trans. Br. Mycol. Soc. 83:487-490, Ferreira et al. (1991) Phytopathology 81:283-287 and Baker et al. (1983) Phytopathology 73:1148-1152. All disclose the use of Bacillus spp. and Bacillus subtilis as biocontrol agents of fungal plant pathogens. Pusey et al. (1988) Plant Dis. 72:622-626, Pusey and Robins (U.S. Pat. No. 5,047,239), and McKeen et al. (1986) Phytopathology 76:136-139 disclose control of post harvest fruit rot using B. subtilis. McKeen et al., supra, have shown that antibiotics similar to the low molecular weight iturin cyclic polypeptides contribute to this fungicidal activity of B. subtilis.
Liu et al. (1995) U.S. Pat. No. 5,403,583 disclose a Bacillus sp., ATCC 55000 and a method to control the fungal plant pathogen, Rhizoctonia solani. Islam and Nandi (1985) J. Plant Dis. Protect. 92(3):241-246, disclose a Bacillus sp. with antagonism to Drechslera oryzae, the causal agent of rice brown spot. The same authors, Islam and Nandi (1985) J. Plant Dis. Protect. 92(3):233-240, also disclose in-vitro antagonism of Bacillus sp. against Drechslera oryzae, Alternaria alternata and Fusarium roseum. They discuss three components in the culture filtrate. The most active antibiotic was highly soluble in water and methanol with a UV peak at 255 nm and a shoulder at 260 mn, that proved to be a polyoxin-like lipopeptide. Cook (1987) Proceedings Beltwide Cotton Production--Mechanization Research Conference, Cotton Council, Memphis, pp. 43-45 discloses the use of a suspension of Bacillus sp. to reduce the number of cotton plants killed by Phymatotrichum omnivorum, a cause of cotton root rot.
B'Chir and Namouchi (1988) (Revue Nematologique 11(2):263-266) report on a Bacillus pumilus that stimulates nematode trapping fungi to increase their ability to trap nematodes. B'Chir and Belkadhi (1986) (Med. Fac. Landbouww. Rijksuniv. Gent 51/3b:1295-1310) discuss the cellular interactions of a fungus (Fusarium) and nematodes that cause infection in citrus. The fungus is associated with B. pumilis (they occur together) and when the nematode is also there, the fungus is more severe. B. pumilus appears to be providing food for the nematodes. Gokte and Swarup (1988) (Indian J. Nematol. 18(2):313-318) report on B. pumilus that are nematicidal, but they do not report any antifungal activity. Slabospitskaya et al. (1992) (Mikrobiol Zh (Kiev) 54(6):16-22) compare many different Bacillus, including B. pumilus for their ability to produce chitinases, but they report no activity on plant pathogens. The B. pumilus produce the lowest chitinase levels. McInroy et al. (1995) Plant and Soil 173(2):337-342, did a survey of the many types of bacteria, including many Bacillus and B. pumilus that are endophytes within plant stems and roots. However, they show no evidence that these endophytic strains are antifungal. Chernin et al. (1995) Molecular Genetics, found a Bacillus pumilus that has a wide spectrum of activity against bacteria (e.g., Xanthomonas, Pseudomonas, Erwinia) and fungi that cause plant disease. Fey et al (1991) Akad Landwirts Kart, report on B. pumilus strains that provide seed potatoes some protection from Rhizoctonia solani.